PCR from blood

neill gingles <nag3 at le.ac.uk> wrote:
>Has anyone any methods or information on screening samples of blood for
>a gene using PCR. Are there any inherant problems involved, such as haem
>inhibition of taq polymerase at certain concentrations, also does the
>type of blood have any effect (for example mouse compared to human). We
>are presently trying to set up a quick screening process using PCR in
>our lab and I would be grateful if anyone has any suggestions.
>Dear Neill,
We have been checking aliquots of mouse blood for a gene knockout.
We use 1µl blood diluted 1:5 in heparin, in a 50µl reaction volume.
[Mg] is 2mM, primers 100ng/µl and Tth 2u/µl, for 35 cycles.
We found Taq not to be reproducible and use Tth.
We always do two reactions for each sample, one with just mouse blood
and the other with mouse blood plus a spike of plasmid DNA carrying
the target template or something similar, adding about 1000 copies to
mimic the genomic target.
If the spiked sample fails to amplify, we know we have a problem with
that blood sample. If it amplifies then we believe the result for the
unspiked blood sample. That level of +ve control is necessary because
blood samples taken from sibs on the same day can behave differently
in our hands.
It is a bit tedious but you can get through a lot of samples.
Rick